The invention provides nucleotide sequences from coryneform bacteria which cod for the histidine kinase luminescence expression sensor (luxS) gene and a process for the fermentative preparation of amino acids using bacteria in which the luxS gene is attenuated.
L-Amino acids, in particular lysine, are used in human medicine and in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition.
It is known that amino that amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and which produce amino acids are obtained in this manner.
Methods of the recombinant DNA technique have also been employed for some years for improving the stain of Corynebacterium strains which produce L-amino acid, by amplifying individual amino acid biosynthesis genes and investigating the effect on the amino acid production.
The inventors had the object of providing new measures for improved fermentative preparation of amino acids.
Where L-amino acids of amino acids are mentioned in the following, this means one or more amino acids, including their salts, chosen from the group consisting of L-glycine, asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanin, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine. Lysine is particularly preferred.
When L-lysine or lysine are mentioned in the following, not only the bases but also the salts, such as e.g. lysine monohydrochloride or lysine sulfate, are meant by this.
The invention provides an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence which codes for the luxS gene, chosen from the group consisting of
a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2,
b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID No. 2,
c) polynucleotide which is complementary to the polynucleotides of a) or b), and
d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c),
the polypeptide preferably having the activity of the histidine kinase LuxS.
The invention also provides the abovementioned polynucleotide, this preferably being a DNA which is capable of replication, comprising:
(i) the nucleotide sequence, shown in SEQ ID No.1, or
(ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or
(iii) at least one sequence which hybridizes with the sequences complementary to sequences (i) or (ii), and optionally
(iv) sense mutations of neutral function in (i).
The invention also provides:
a polynucleotide, in particular DNA, which is capable of replication and comprises the nucleotide sequence as shown in SEQ ID No.1;
a polynucleotide which codes for a polypeptide which comprises the amino acid sequence as shown in SEQ ID No.2;
a vector containing parts of the polynucleotide according to the invention, but at least 15 successive nucleotides of the sequence claimed,
and coryneform bacteria in which the luxS gene is attenuated, in particular by an insertion or deletion.
The invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library of a coryneform bacterium, which comprises the complete gene or parts thereof, with a probe which comprises the sequence of the polynucleotide according to the invention according to SEQ ID No.1 or a fragment thereof, and isolation of the polynucleotide sequence mentioned.
Polynucleotides which comprises the sequence according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate, in the full length, nucleic acids or polynucleotides or genes which code for the histidine kinase LuxS or to isolate those nucleic acids or polynucleotides or genes which have a high similarity with the sequence the luxS gene.
Polynucleotides which comprises the sequences according to the invention are furthermore suitable are primers with the aid of which DNA of genes which code for the histidine kinase LuxS can be prepared by the polymerase chain reaction (PCR).
Such oligonucleotides which serve as probes or primers comprise at least 30, preferably at least 20, very particularly preferably at least 15 successive nucleotides. Oligonucleotides which have a length of at least 40 or 50 nucleotides are also suitable.
FIG. 1: Map of the plasmid pCR2.1 luxSint.
The abbreviations and designations used have the following meaning.
KmR: Kanamycin resistance gene
EcoRI: Cleavage site of the restriction enzyme EcoRI
PstI: Cleavage site of the restriction enzyme PstI
SalI: Cleavage site of the restriction enzyme SalI
luxSint: Internal fragment of the luxS gene
ColE1: Replication origin of the plasmid ColE1.
xe2x80x9cIsolatedxe2x80x9d means separated out of its natural environment.